Detection of microbial disturbances in a drinking water microbial community through continuous acquisition and advanced analysis of flow cytometry data.

Detecting disturbances in microbial communities is an important aspect of managing natural and engineered microbial communities. Here, we implemented a custom-built continuous staining device in combination with real-time flow cytometry (RT-FCM) data acquisition, which, combined with advanced FCM fingerprinting methods, presents a powerful new approach to track and quantify disturbances in aquatic microbial communities. Through this new approach we were able to resolve various natural community and single-species microbial contaminations in a flow-through drinking water reactor. Next to conventional FCM metrics, we applied metrics from a recently developed fingerprinting technique in order to gain additional insight into the microbial dynamics during these contamination events. Importantly, we found that multiple community FCM metrics based on different statistical approaches were required to fully characterize all contaminations. Furthermore we found that for accurate cell concentration measurements and accurate inference from the FCM metrics (coefficient of variation ≤ 5%), at least 1000 cells should be measured, which makes the achievable temporal resolution a function of the prevalent bacterial concentration in the system-of-interest. The integrated RT-FCM acquisition and analysis approach presented herein provides a considerable improvement in the temporal resolution by which microbial disturbances can be observed and simultaneously provides a multi-faceted toolset to characterize such disturbances.

Chelativorans multitrophicus gen. nov., sp. nov. and Chelativorans oligotrophicus sp. nov., aerobic EDTA-degrading bacteria.

Two previously isolated strains (DSM 9103(T) and LPM-4(T)) able to grow with EDTA (facultatively and obligately, respectively) as the source of carbon, nitrogen and energy were investigated in order to clarify their taxonomic positions. The strains were strictly aerobic, Gram-negative, asporogenous and non-motile rods that required biotin for growth. Reproduction occurred by binary fission. The strains were mesophilic and neutrophilic. Their major fatty acids were summed feature 7 (consisting of C(18 : 1)omega7c, C(18 : 1)omega9t and/or C(18 : 1)omega12t) and C(19 : 0) cyclo omega8c. The polyamine pattern revealed homospermidine as a major polyamine. Predominant polar lipids were phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine, phosphatidyldimethylethanolamine, phosphatidylmonomethylethanolamine and diphosphatidylglycerol. Mesorhizobium-specific ornithine lipid was absent. The predominant isoprenoid quinone was Q-10. The DNA G+C values were 60.8 and 63.1 mol% (T(m)) for strains LPM-4(T) and DSM 9103(T), respectively. The level of 16S rRNA gene sequence similarity between these EDTA-utilizers was 99.3 % while the DNA-DNA hybridization value was only 37 %. Both strains were phylogenetically related to members of the genera Aminobacter and Mesorhizobium (95-97 % sequence similarity). However, DNA-DNA hybridization values between the novel EDTA-degrading strains and Aminobacter aminovorans DSM 7048(T) and Mesorhizobium loti DSM 2626(T) were low (10-11 %). Based on their genomic and phenotypic properties, the new alphaproteobacterial strains are assigned to a novel genus, Chelativorans gen. nov., with the names Chelativorans multitrophicus sp. nov. (type strain DSM 9103(T)=VKM B-2394(T)) and Chelativorans oligotrophicus sp. nov. (type strain LPM-4(T)=VKM B-2395(T)=DSM 19276(T)).

The apparent clock-like evolution of Escherichia coli in glucose-limited chemostats is reproducible at large but not at small population sizes and can be explained with Monod kinetics.

To follow and model evolution of a microbial population in the chemostat, parameters are needed that give an indication of the absolute extent of evolution at a high resolution of time. In this study the evolution of the maximum specific growth rate ( micro (max)) and the residual glucose concentration was followed for populations of Escherichia coli K-12 under glucose-limited conditions at dilution rates of 0.1 x h(-1), 0.3 x h(-1) and 0.53 x h(-1) during 500-700 h in continuous culture. Whereas micro (max) improved only during the initial 150 h, the residual glucose concentration decreased constantly during 500 h of cultivation and therefore served as a convenient parameter to monitor the evolution of a population at a high time resolution with respect to its affinity for the growth-limiting substrate. The evolution of residual glucose concentrations was reproducible in independent chemostats with a population size of 10(11) cells, whereas no reproducibility was found in chemostats containing 10(7) cells. A model based on Monod kinetics assuming successive take-overs of mutants with improved kinetic parameters (primarily K(s)) was able to simulate the experimentally observed evolution of residual glucose concentrations. Similar values for the increase in glucose affinity of mutant phenotypes (K(s(mutant)) approximately equal 0.6 x K(s(parent))) and similar mutation rates per cell per generation leading to these mutant phenotypes (1-5 x1 0(-7)) were estimated in silico for all dilution rates. The model predicts a maximum rate of evolution at a dilution rate slightly below micro (max)/2. With increasing and decreasing dilution rates the evolution slows down, which also explains why in special cases a selection-driven evolution can exhibit apparent clock-like behaviour. The glucose affinity for WT cells was dependent on the dilution rate with highest values at dilution rates around micro (max)/2. Below 0.3 x h(-1) poorer affinity was mainly due to the effects of rpoS.

Isolation and growth characteristics of an EDTA-degrading member of the alpha-subclass of Proteobacteria.

A Gram-negative, ethylenediaminetetraacetic acid (EDTA)-degrading bacterium (deposited at the German Culture Collection as strain DSM 9103) utilising EDTA as the only source of carbon, energy and nitrogen was isolated from a mixed EDTA-degrading population that was originally enriched in a column system from a mixture of activated sludge and soil. Chemotaxonomic analysis of quinones, polar lipids and fatty acids allowed allocation of the isolate to the alpha-subclass of Proteobacteria. 16S rDNA sequencing and phylogenetic analysis revealed highest similarity to the Mesorhizobium genus followed by the Aminobacter genus. However, the EDTA-degrading strain apparently forms a new branch within the Phyllobacteriaceae/Mesorhizobia family. Growth of the strain was rather slow not only on EDTA (micro(max) = 0.05 h(-1)) but also on other substrates. Classical substrate utilisation testing in batch culture suggested a quite restricted carbon source spectrum with only lactate, glutamate, and complexing agents chemically related to EDTA (nitrilotriacetate, iminodiacetate and ethylenediaminedisuccinate) supporting growth. However, when EDTA-limited continuous cultures of strain DSM 9103 were pulsed with fumarate, succinate, glucose or acetate, these substrates were assimilated immediately. Apparently, the strain can use a broader spectrum than indicated by traditional substrate testing techniques. The EDTA species CaEDTA and MgEDTA served as growth substrates of the strain because in the mineral medium employed EDTA was predicted to be mainly present in the form of these two complexes. The bacterium was not able to degrade Fe3+-complexed EDTA.